Most bacterial ORFs are identified by automated prediction algorithms. However, these algorithms often fail to identify ORFs lacking canonical features such as a length of >50 codons or the presence of an upstream Shine-Dalgarno sequence. Here, we use ribosome profiling approaches to identify actively translated ORFs in Mycobacterium tuberculosis. Most of the ORFs we identify have not been previously described, indicating that the M. tuberculosis transcriptome is pervasively translated. The newly described ORFs are predominantly short, with many encoding proteins of ≤50 amino acids. Codon usage of the newly discovered ORFs suggests that most have not been subject to purifying selection, and hence are unlikely to contribute to cell fitness. Nevertheless, we identify 90 new ORFs (median length of 52 codons) that bear the hallmarks of purifying selection. Thus, our data suggest that pervasive translation of short ORFs in Mycobacterium tuberculosis serves as a rich source for the evolution of new functional proteins.
Keywords: Mycobacterium tuberculosis; infectious disease; leaderless; microbiology; none; pervasive translation; sORF; small protein.
How can you predict which proteins an organism can make? To answer this question, scientists often use computer programs that can scan the genetic information of a species for open reading frames – a type of DNA sequence that codes for a protein. However, very short genes and overlapping genes are often missed through these searches. Mycobacteria are a group of bacteria that includes the species Mycobacterium tuberculosis, which causes tuberculosis. Previous work has predicted several thousand open reading frames for M. tuberculosis, but Smith et al. decided to use a different approach to determine whether there could be more. They focused on ribosomes, the cellular structures that assemble a specific protein by reading the instructions provided by the corresponding gene. Examining the sections of genetic code that ribosomes were processing in M. tuberculosis uncovered hundreds of new open reading frames, most of which carried the instructions to make very short proteins. A closer look suggested that only 90 of these proteins were likely to have a useful role in the life of the bacteria, which could open new doors in tuberculosis research. The rest of the sequences showed no evidence of having evolved a useful job, yet they were still manufactured by the mycobacteria. This pervasive production could play a role in helping the bacteria adapt to quickly changing environments by evolving new, functional proteins.